Thesis Reference

Thesis Targeting Waddlia chondrophila development cycle using genetic tools and metabolomics WALTER, Chantal Abstract All members of the Chlamydiae, including Waddlia chondrophila share the same unique intracellular development cycle, which is the focus of this work in the search for anti-chlamydial targets and drugs. Strategies using high throughput screening, genetic complementation, and metabolomics were explored to investigate and try to block the W. chondrophila development cycle. First, a generic high throughput screening procedure was developed in a heterologous system, Escherichia coli, to search for inhibitors of chlamydial transcription factors. Then, the function of DksA, a transcription factor, was investigated by genetic complementation of E. coli and Pseudomonas aeruginosa mutants. Finally, metabolomics was adopted to observe changes in metabolites associated with progress through the chlamydial developmental cycle. It revealed the presence of putative novel metabolites differentially expressed in the various stages. Altogether, the results of this work present putative new therapeutic approaches to understand and circumvent the infection process of this pathogen. Reference WALTER, Chantal. Targeting Waddlia chondrophila development cycle using genetic tools and metabolomics. Thèse de doctorat : Univ. Genève, 2019, no. Sc. 5337 DOI : 10.13097/archive-ouverte/unige:119059 URN : urn:nbn:ch:unige-1190597 Available at: http://archive-ouverte.unige.ch/unige:119059 Disclaimer: layout of this document may differ from the published version. 1 / 1 UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section des Sciences Pharmaceutiques Professeur Muriel Cuendet Laboratoire de Pharmacognosie Docteur Philippe Christen Département de Botanique et Biologie Végétale Docteur Karl Perron ________________________________________________________________________ Targeting Waddlia chondrophila development cycle using genetic tools and metabolomics THÈSE Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention sciences pharmaceutiques Par Chantal Walter de Mümliswil-Ramiswil (SO) Thèse N° 5337 GENÈVE Atelier d’impression ReproMail 2019 I. Abstract Chlamydia sp. are the major cause of bacterial eye infection in the world. They are also responsible for several urogenital infections. The consequences of such untreated infections can be blindness and infertility. According to the World Health Organization, more than 131 million people were newly infected in 2016 and Chlamydia infections in the world’s population is still on the rise. At the moment, their treatment is based on antibiotics such as macrolides and tetracyclines. However, since general antibiotic resistance and frequency of persistent infections in the world are rapidly increasing, it is necessary to develop new strategies to treat these bacterial infections. These human pathogens are all Gram-negative intracellular bacteria and members of the phylum Chlamydiae. The Chlamydiae member Waddlia chondrophila is especially interesting as a model organism since it shares the same intracellular development cycle as the other members of the phylum, but is less pathogenic. The biphasic development cycle comprises the infectious form, called the elementary body (EB) and the non-infectious metabolically active form, the reticulate body (RB). This developmental cycle is the focus of the work presented here. It is unique to the Chlamydiae and thus is of interest in the search for novel anti-chlamydial targets and drugs. Patterns of gene expression, which are up- or down-regulated during this development cycle were identified and in some cases this temporal gene expression has been linked to the activity of specific transcription factors (TF). There are relatively few TFs within the Chlamydiae, and only eight are common throughout the phylum. These latter appear to be important for progress through the developmental cycle. The W. chondrophila TF Euo is a major regulator of the late phase gene expression when the differentiation from RBs to EBs occurs. It is found only in the Chlamydiae and thus was chosen as a target in the search for specific inhibitors. Since no experimental genetic system is currently available for studying Chlamydiae, and manipulating the Chlamydiae genome to create specific mutations has not been possible thus far, a high throughput screening procedure was developed in a heterologous system, Escherichia coli. For these experiments, the W. chondrophila Euo was co-expressed with a reporter plasmid containing a known Euo promoter fused to β-galactosidase (LacZ). This made it possible to measure Euo activity in an experimentally tractable host, without the need to culture the Chlamydia cells. Natural products encompass a wide range of chemically diverse molecules and in the past have also proved to be a rich source of potential antibiotics. In this study, a total of around 175 plant extracts and 2700 natural products were screened against of Euo. The method proved suitable for use in high-throughput screening mode although unfortunately no active compound emerged. -I- A different strategy was therefore adopted focusing on another chlamydial TF, DksA. Unlike Euo, DksA is present in a wide range of bacteria including E. coli and Pseudomonas aeruginosa. This suggested the possibility of investigating W. chondrophila DksA function by genetic complementation of DksA mutants of E. coli and P. aeruginosa, with the subsequent aim of developing a screening protocol to identify W. chondrophila DksA inhibitors. DksA is a DNA-independent TF which binds to RNA polymerase through the so-called secondary channel. In E. coli, DksA is known to be a key regulator of the stringent response to unfavourable growth conditions, such as amino acid starvation, iron limitation or changes in pH of the environment. Its major effects on gene expression are down-regulation of rRNA transcription and up-regulation of genes required for synthesis of several amino acids. In P. aeruginosa, DksA is known to be involved in production of pyocyanin, an important virulence factor. For the complementation experiments, W. chondrophila dksA was transformed into dksA mutant strains of E. coli and P. aeruginosa and tested for its ability to complement the host cell mutation. However, no restoration of DksA function in either E. coli or P. aeruginosa DksA was observed in any of these tests. Given the difficulties encountered in studying the function of recombinant chlamydial TFs in a heterologous host, a very different approach was adopted in which changes in the metabolites associated with progress through the chlamydial developmental cycle were investigated in a mammalian cell host. For these experiments, the Vero monkey kidney epithelial cells were infected with W. chondrophila and the cellular metabolites were monitored at early and late stage of the development cycle, corresponding to the transition from the RB stage to the final EB stage of infection. The results obtained using ultra-high performance liquid chromatography time-of-flight mass spectrometry revealed three metabolites which were increased during the late stage of the developmental cycle, and which were not present in non-infected cells. Molecular networks generated from the tandem mass spectra (MS2) showed a cluster of the three increased masses, although they could not be identified by dereplication, probably due to the lack of an extensive database of Chlamydiae metabolites. According to MS 2 and high resolution mass spectra data, these molecules are probably lipids. Indeed, the MS2 spectra of all three compounds showed a fragmentation ion which could tentatively be assigned to cholesterol with a loss of the alcohol group as water. The network clustering, the MS 2 spectra and the high resolution mass spectra of these three metabolites produced in W. chondrophila- infected Vero cells suggest that they are as yet unknown metabolites, with a cholesteryl ester structure and a diamide side chain. In conclusion, different approaches were used in this work with the aim of understanding the biology of the chlamydial infectious cycle, and ultimately of identifying new targets and anti- chlamydial drugs. Although, the screening of natural products yielded no significant positive -II- results, this research provides a generic screening tool, which could be used to search for inhibitors of other chlamydial TFs. The complementation approach using the chlamydial TF DksA, expressed in the corresponding mutants of two other Gram-negative bacteria, E. coli and P. aeruginosa, showed no complementation, thus suggesting differences in the regulation and/or function of DksA between these diverse bacterial species. Finally, the metabolomics research revealed the presence of putative new metabolites characteristic of different stages during the development cycle. These metabolites are probably unique, and thus could be characteristic for W. chondrophila infections. Overall, this work presents several strategies using high throughput screening, genetic complementation, and metabolomics to investigate the W. chondrophila development cycle. While targeting specific TFs did not yield useful results, the findings of the metabolomic study has provided novel insight into the W. chondrophila development cycle, which in the future could offer a promising starting point for identifying specific and druggable targets to perturb the chlamydial infectious cycle. -III- -IV- II. Résumé Les bactéries du genre Chlamydia sont la

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